The invention relates generally to multicellular tissue-like compositions.
Tissue equivalents are three-dimensional living multicellular tissue-like compositions. While these tissue equivalents have many uses, including tissue transplantation, screening and evaluation of new drugs, previous tissue equivalents have had limited utility because they contracted. For example, a tissue equivalent comprising a collagen matrix can exhibit as much as about 80% linear shrinkage, i.e., can contract to as little as twenty percent of the original diameter within a period of a few hours. This contraction produces a dense, opaque matrix which prevents the visualization of the contained cells by optical microscopy. The resulting tissue equivalent may resemble normal scar tissue more than the desired normal healthy tissue. The factors responsible for such contraction have not been systematically evaluated and studied, but may include collagen concentration and cell numbers.
In general, tissue equivalents are produced by combining at least one cellular component with at least one noncellular component. The design and construction of tissue equivalents is a branch of tissue engineering, which can be defined as the application of scientific principles to the design, construction modification, growth and maintenance of living tissues to form the desired composition.
Tissue-equivalents have numerous uses including: sources of tissue for transplantation; systems for screening and evaluating potential drugs, cosmetics and other consumer products; model systems for the study of multicellular processes such as wound healing; systems for establishing optimal conditions for trans-tissue delivery of hormones, cytokines or other biologically active materials and systems for introducing cells genetically engineered to produce a desired substance. It would be desirable to use such tissue equivalents to decrease dependency on cadaver tissue for grafts and transplants and to reduce dependency on animal testing in the development of new pharmaceuticals and consumer products.
xe2x80x9cTissue equivalentxe2x80x9d as used herein includes, but is not limited to, artificially produced epithelial tissue, skin, cornea, connective tissue, cartilage, bone, and the like (see for example, U.S. Pat. Nos. 4,485,096; 4,485,097; 4,546,500; 4,539,716; 4,604,346; 4,835,102).
The cellular component of tissue equivalents may be derived from a number of sources. The cells comprising the cellular component may be autologous, that is, the donor and the recipient may be the same person. The cells are processed, incorporated into the non-contracting tissue equivalent, and transplanted back into the donor as part of the tissue equivalent. Alternatively, the cells may be allogenic, that is taken from a different donor than the recipient of the transplanted tissue equivalent, where both the donor and recipient are members of the same species. The cells also may be xenogeneic, i.e., derived from a donor of a different species from the recipient. In each of these cases, treatments are known in the art that reduce the likelihood of rejection or control the differentiation of the cellular component. Human cells, i.e., either autologous or allogenic cells, are preferred.
The noncellular component of tissue equivalents may comprise one or more of a group of compounds, including compounds normally secreted by cells to form a naturally occurring extracellular matrix. Suitable compounds include the collagens.
The collagens are a family of fibrous proteins that are secreted by connective tissue cells, as well as by a variety of other cell types. See generally, Alberts, B., et al., Molecular Biology of the Cell, 3rd Ed., Garland Publishing, New York (1994) pp. 978-984. The characteristic feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains, called xcex1 chains, are wound around one another in a rope-like superhelix. About 25 distinct collagen xcex1 chains have been identified, each encoded by a separate gene.
About fifteen different types of collagen have been described, which are characteristically composed of different combinations of specific xcex1 chains. Type I collagen (collagen I) is the principal collagen of skin, tendon, ligaments, cornea, internal organs and bone. Collagen I is by far the most common, accounting for about 90% of body collagen. The xcex1 chain composition of collagen I is [xcex11(I)]2xcex12(I).
Other fibrillar collagens are types II, III, V, VII and XI. Type II collagen (collagen II) cartilage, composed of [xcex11(II)]3 xcex1 chains, is found in cartilage, the intervertebral discs of the spine and the vitreous humor of the eye. Type III collagen (collagen III), [xcex11(III)]3, is found in skin, blood vessels and internal organs. Type V collagen (collagen V), [xcex11(V)]2xcex12(V), is found in the same tissues as type I collagen. Type XI collagen (collagen XI), xcex11(XI)xcex12(XI)xcex13(XI), is found in the same tissues as collagen I. Alberts, et al., page 980.
In contrast to the above fibrillar collagens, network-forming collagens form a felt-like sheet or meshwork instead of rope-like fibers. An important network-forming collagen is collagen IV, [xcex11(IV)]2xcex12(IV), which forms the basal lamina. The basal lamina, sometimes called the basement membrane, is a thin mat of extracellular matrix that separates the epithelium from the underlying stroma/connective tissue. The basal lamina also separates many other types of cells, such as muscle cells and fat cells, from connective tissue.
In previous tissue equivalents, for example those described in Clark et al., J. Clin. Invest. 84: 1036-1040 (1989) and Montesano et al., Proc. Nat. Acad. Sci. U.S.A. 85: 4894-4897 (1988), the collagen matrix contracts after formation to a fraction of its original size (typically to about twenty percent of the original diameter) over a period of up to 48 hours. The contraction of the tissue equivalent as a whole follows the contraction of the collagen matrix. As a result, the matrix condenses forming a dense, opaque tissue which prevents visualization of the contained cells by transmitted light or fluorescence microscopy. The multiple factors responsible for contractions have not been studied systematically but it has been proposed that they include cell number and collagen concentration. In addition, unknown combinations of cytokines, such as presumably present in exogenously supplied serum such as fetal bovine serum (FBS) may be responsible for contraction.
Contraction of tissue equivalents may be desirable for some limited number of uses, for example, in wound closure or scar formation. However, extensive contraction produces an abnormally dense scar-like tissue that impedes normal tissue functions such as epithelialization, vascularization, pigmentation and hair growth. Contraction of tissue equivalents is thus a problem for which a solution has been sought for several years.
Previous non-contracting tissue equivalents have been constructed using pre-formed collagen sponge matrices. Collagen sponge matrices are composed of insoluble, covalently-linked, solid collagen fibrils. Covalent cross-links are formed between collagen fibrils by chemical reactions and thus cannot be readily reversed. The physical form of collagen sponges produced by chemical cross-linking can only be altered by digestion with collagenase, an enzyme which degrades collagen into its component amino acids. In addition, collagen sponges may retain the toxic chemical reagents used in cross-linking, such as aldehydes, which may leach into the host tissues, causing adverse reactions.
What is needed is a non-contracting tissue equivalent that provides dimensional stability and permits the monitoring of the functions of viability of the cellular component.
The present invention provides a method for producing a substantially non-contracting tissue equivalent comprising a three-dimensional, dimensionally substantially stable collagenous matrix populated by mesenchymal cells, which cells can be from a variety of sources. The collagenous matrix is substantially stable dimensionally and does not contract, i.e., does not substantially change in wet weight, volume and density, for at least twenty one days. The substantially non-contracting tissue equivalent of the present invention can be maintained in vitro for at least six months.
The tissue equivalent so produced comprises at least one non-cellular component and at least one cellular component. Suitable non-cellular components are naturally occurring collagenous materials such as collagen I, collagen III, collagen IV, hyaluronic acid, chitosan, chondroitin-6 sulfate, fibrin, fibronectin, and mixtures thereof. Collagenous materials suitable as matrix components preferably are chosen from the group consisting of collagen I, collagen III, collagen IV, fibrin, fibronectin and mixtures thereof. One particularly preferred matrix component is collagen I.
The non-cellular component can also include synthetic materials in addition to, or instead of, the collagenous materials. Suitable such synthetic materials include polyglycolic acid, polylactic acid, polyhydroxybutyrate, and the like.
Preferably the tissue equivalents of the present invention have a non-cellular component that is a collagenous matrix and a cellular component that comprises mesenchymal cells such as fibroblasts, and the like. Typically a mixture of collagen and fibroblasts is allowed to thicken, preferably by the gelation (fibrillogenesis) of the collagen.
Suitable cellular components are mesenchymal cells selected from cells of multicellular animals, preferably from mammalian cells, and are optimally human cells. The human cells may be autologous, that is, derived from the same individual who will ultimately receive a graft of the tissue equivalent. Alternatively, cells may be selected that originate from another human or from individuals of another species. If non-autologous cells are used, appropriate means of suppressing any immune response to the tissue equivalent are known to one of ordinary skill.
Suitable are mesenchymal cells which may originate from a variety of tissues and are chosen preferably from the group consisting of fibroblasts, keratinocytes, melanocytes and mixtures thereof. One such preferred cellular component is human fibroblasts.
A three-dimensional tissue equivalent can be formed by a method comprising the steps of combining an aqueous suspension of at least one mesenchymal cell type with at least one soluble collagenous material and gelling the soluble collagenous material to form a substantially non-contracting tissue equivalent constituted by a collagenous matrix with mesenchymal cells. Preferably the soluble collagenous component is collagen I, which gels at about pH 7 upon warming to about body temperature to form a substantially non-contracting translucent matrix. This translucent matrix is hydrophilic, free from covalent crosslinks, and can be liquified in an acidic pH environment.
The basic three-dimensional tissue equivalent can serve as the foundation for the construction of more complex products comprising additional cellular components and as well as collagenous components. The basic three-dimensional tissue equivalent is produced by a method wherein an aqueous suspension of mesenchymal cells in a nutrient medium is combined with at least one soluble collagenous component at a temperature below about ambient temperature and the resulting admixture is solidified by gelation at about 37xc2x0 C. and at a pH of about 7 to a translucent matrix that contains mesenchymal cells. A further collagenous material and additional mesenchymal cells can be added to form a substantially non-contracting multicellular tissue equivalent, if desired.
In one embodiment, the first cellular component comprises fibroblasts, a first soluble extracellular matrix component comprises collagen I, a second cellular component comprises keratinocytes, and a second soluble extracellular matrix component comprises collagen IV. In another embodiment, a first cellular component comprises corneal fibroblasts, e.g., keratocytes, and a first soluble extracellular matrix component comprises collagen I, a second cellular component comprises corneal epithelial cells, and a second soluble extracellular matrix component comprises collagen IV. In a further embodiment, a first cellular component comprises corneal fibroblasts such as keratocytes and a first soluble extracellular matrix component comprises collagen I, a second cellular component comprises corneal endothelial cells, and a second soluble extracellular matrix component comprises mixture of collagen I, fibronectin, and laminin.
Additional compatible cellular components and non-cellular components may also be used in forming the tissue equivalent. For example, a tricellular three-dimensional tissue equivalent is produced by contacting an aqueous suspension of a first cellular component with at least one soluble collagenous matrix component; dispersing the first cellular component in the soluble matrix component; and gelling the resulting admixture to form a dermal equivalent having a first or top surface and a second or bottom surface. The top surface of the dermal equivalent is then contacted with a solution of another cellular component, and optionally with extracellular matrix components, to form a layer thereof which upon gelling forms a substantially non-contracting bicellular three-dimensional tissue equivalent. The non-contracting bicellular three-dimensional tissue equivalent is then similarly contacted with another set of cellular and extracellular components to form a tricellular, three-dimensional tissue equivalent. In one illustrative embodiment, the initial cellular component comprises fibroblasts, and the extracellular collagenous matrix component therefor comprises collagen, another cellular component comprises melanocytes, another soluble extracellular collagenous matrix component comprises collagen IV, and a further cellular component comprises keratinocytes.
The substantially non-contractile characteristic of this tissue equivalent is independent of cell density in the range of about 1.0xc3x97105 to about 5.0xc3x97105 cells/ml, and is independent of collagen concentration in the range of about 3 to about 5 mg/ml. The cells which are used to establish the non-contracting tissue equivalent may be from any passage but early passage cells (up to passage 5) are preferred, and may be taken from donors of any age and sex. Cells from the skin of young donors (e.g., infant) are preferred, however.
The substantially non-contractile quality of the tissue equivalent is characterized by the lack of substantial change in wet weight, volume and density over time. More specifically, the non-contractile quality of the tissue equivalent is characterized by less than about 5% shrinkage over a period of about twenty-one days. An additional important unique advantage of the non-contracting tissue equivalent is its translucency, which allows direct visual observation of its component cells by optical microscopy.
The non-contracting tissue equivalent provided by the present invention more closely resembles normal tissue than any tissue equivalent previously described. The cellular component of this tissue equivalent is quiescent until stimulated. Appropriate stimuli can induce the non-contracting tissue equivalent to undergo cell division, synthesize extracellular matrix macromolecules, migration of cells, or undergo contraction. The non-contracting tissue equivalent can support growth and differentiation of epithelial cells as well as the growth of endothelial cells. Both the epithelial and the endothelial surfaces thus produced on the non-contracting tissue equivalent display characteristic histological features of normal tissues.
The non-contracting tissue equivalent is hydrophilic and translucent, permitting the visual observation of the cellular components by transmitted light and fluorescence microscopy. Cellular viability, cell motility, as well as cellular growth and differentiation can be directly observed. Thus, quantitative evaluation of the status of cells of the non-contracting tissue equivalent can be conveniently and rapidly assessed by either manual or automated methods.
In contrast to the contracting tissue equivalents that lose water, resulting in the condensation of the matrix, equivalent to the formation of a scar, the matrix of the non-contracting tissue equivalent remains substantially hydrated, and thus maintains a greater natural permeability to exogenous materials such as nutrients or drugs. This greater natural permeability of the non-contracting tissue equivalent also provides a more realistic system in which to study the processes of tissue contraction and consequent scarring. Thus, the non-contracting tissue equivalent of the present invention provides a useful system for the study of fundamental mechanisms and therapeutic approaches in wound healing.
The non-contracting tissue equivalent of the present invention is generally useful in supporting the growth and differentiation of various epithelial and endothelial cells. The non-contracting tissue equivalent, when used as a support for epithelial cells, can support cellular differentiation without the use of exogenous agents, such as retinoic acid.
The non-contracting tissue equivalent can be used as a transplant material, providing a barrier that assists the recipient in maintaining proper hydration, excluding pathogens and assisting thermoregulation. Mechanically, the non-contracting tissue equivalent is robust enough to survive manual manipulation.
The non-contracting tissue equivalent can also be used as a system for screening of potential drugs and consumer products. This tissue equivalent can be used to test substances administered in either a systemic mode (test substance applied to the endothelial side) or a topical mode (test substance applied to the epithelial side).
The non-contracting tissue equivalent can also be used as an implantable source of exogenous substances, such as substances used to facilitate processes such as wound healing. Cell types that naturally secrete useful substances, such as cytokines, can be incorporated as part or all of the cellular component of the tissue equivalent. Alternatively, cells such as fibroblasts that have been genetically engineered to enhance normal expression of a product or to express a recombinant protein, can be incorporated as part or all of the cellular component of the tissue equivalent.
The non-contracting tissue equivalents are also useful for studies of the effects of drugs, cosmetics and other pharmaceutical agents by more invasive methods. For example, following exposure to various agents, the non-contracting tissue equivalents may be frozen, embedded in a suitable embedding composition and sectioned for histochemical determination of cellular or extracellular enzymatic activities, and peptide and protein functionality. Alternatively, the tissue equivalents may be fixed, embedded in paraffin or other suitable embedding composition and sectioned for examination using optical microscopy. Histochemical, immunohistochemical, and immunofluorescent methods to establish the presence of absence of specific proteins, glycoproteins, and proteoglycans may be used on frozen sections or sections that have been treated to remove the matrix of embedding compound. Sections of the non-contracting tissue equivalent can also be used to assess gene expression by in situ hybridization with nucleotide probes complementary to specific nucleic acid sequences.
The fixed tissue equivalents may also be embedded in plastic resins, thin-sectioned and observed using transmission electron microscopy for evaluation of ultrastructural changes. Several techniques permit one skilled in the art to visualize antibody or oligonucleotide probes. For example, treatment of the tissue with gold labeled antibodies (immunogold labeling) can visualize antibodies to specific proteins, glycoproteins, and proteoglycans and precisely delineate minute changes in their localization or quantities. Similar precision in monitoring nuclear events can be attained by using labeled oligoriboprobes. Gold, alone or with silver enhancement, may be localized in the cells and tissues because it is opaque to the beam bombarding the specimen. Surfaces of the fixed tissues may be coated with osmium and examined by scanning electron microscopy because it is opaque to the beam bombarding the specimen. Such techniques allow evaluation of changes in the ultrastructural features of the surface of the tissue equivalent caused by the exposure to a variety of environments or materials.
In addition to the above methods for directly assessing the effects of external conditions on the tissue equivalents, the individual constituents of the tissue equivalent can be dissociated and the individual components thereof isolated and analyzed separately. For example, the collagenous matrix of the tissue equivalent can be dissociated by mild treatment with collagenase. This also is much more accurate and reproducible on a non-contracting tissue equivalent than on a contracted, scar-like matrix. The cell numbers can be determined by usual methods, e.g., using a hemocytometer or flow cytometry.
Cellular and extracellular changes in molecular composition can be quantitated by analytical biochemical or molecular biology methods. Cellular and molecular processes can also be labeled, radioisotopically or otherwise to increase sensitivity of methods for quantitative or qualitative analysis.
The status of the cellular and extracellular components of the non-contracting connective tissue equivalents can be readily evaluated since they are translucent. These unique features allow direct visual observation of the cellular component of the tissue equivalents by light microscopy. If the cells are labeled with a vital dye such as neutral red, an inclusion viability dye, and exposed to different agents, optical microscopic evaluation allows a qualitative picture of the consequences. In this example, neutral red staining demonstrates cellular viability and failure to take up neutral red would indicate toxicity. Due to the translucency of the non-contracting tissue equivalent, it is possible to scan an optical field and determine the number of total cells and the number of viable cells. This procedure can be conducted either manually or by automated scanners. By scanning the matrix in three dimensions, changes in the cell number or cellular orientation can be determined. The present invention thus provides a rapid method for assessing agents which may be toxic or otherwise change the physiological status of a tissue.
Similarly, as seen in FIG. 5, confocal microscopy with computer assisted image processing can be used to quantitate changes in the numbers of viable cells, if the cells are labeled with suitable fluorescent markers. Cell Tracker(trademark) Orange, 5-(and 6-)-(((4-chloromethyl)benzyol)amino)-tetramethylrhodamine (Molecular Probes, Eugene, Oreg.), is a preferred fluorescent reagent to demonstrate viable cells, since only living cells possess the intracellular enzymes required to convert the reagent to the fluorescent product that is detected. Labeling cells with fluorescent markers also permits the observation of changes in the orientation or migration of the cells in addition to determining viability. When a suitable mechanical stage and a suitable microscopy system are used, automated assessment of cell viability and growth can be monitored easily due to the translucent nature of the non-contracting tissue equivalent.
The thickness of the tissue equivalent may impose a limit of this methodology, since the ability to detect labeled cells is impaired by background autofluorescence that increases with thickness. However, single/double photon confocal microscopy can overcome the problem of background autofluorescence. In single/multiple photon confocal microscopy, the tissue is scanned by a laser that only excites the fluorescent marker and background autofluorescence in the plane of the scan, thereby forming optical sections and increasing the effective brightness of the labeled cell compared to the background autofluorescence. Multiple photon evaluation utilizing cells"" innate fluorescence, e.g., that due to the NAD/NADH system can be utilized as well. The latter approach could be applied to living tissue equivalents or to organisms in vivo without having to prelable fluorescently the cells.
The translucence of the non-contracting tissue equivalent facilitates types of monitoring that support spectroscopic analyses. The non-contracting tissue equivalent also is ideal for other minimally invasive methods, such as, studies of metabolic processes using nuclear magnetic resonance (nmr) spectroscopy and metabolic substrates labeled with paramagnetic stable isotopes. Natural abundance of paramagnetic stable isotopes can also be used for monitoring processes of interest.
The non-cellular component and the incorporated cellular component may remain attached to the vessel in which it was formed or it may detach from the vessel and float in the culture medium. In the latter case, these tissue equivalents are referred to as xe2x80x9cfloating culturesxe2x80x9d or xe2x80x9craft cultures.xe2x80x9d